The overall goal of this project is to achieve long-term expression of a desired gene in vivo in a significant percentage of keratinocytes by identifying and introducing genes into keratinocyte stem cells (KSC). We have developed an skin gene therapy model in which human keratinocytes are engineered to express atrial natriuretic peptide (ANP) in human keratinocytes as an approach to effectively treat systemic hypertension. ANP has both natriuretic (or diuretic) and vasorelaxant effects and generally counterbalances the effects of the renin-angiotensin-aldosterone system. Our success in this hypertension model will now allow us to pursue other therapeutic strategies with skin gene therapy. Achieving long-term expression of desired genes such as ANP in a significant percentage of keratinocytes has proven to be very difficult because unique cell surface markers for KSC are not yet known, making the isolation, purification, and efficient introduction of genes into these cells difficult. In order to achieve long-term expression, we have developed an approach using topical colchicine treatments to select for keratinocytes that express a multi-drug resistance (MDR) selectable marker gene along with a linked gene of interest, such as ANP. A significant advantage of this approach is that it does not depend on keratinocyte stem cell identification and targeting. When the selective treatment (colchicine) is applied topically, only keratinocytes that express the MDR selectable marker gene will survive and populate the epidermis. We have recently demonstrated the feasibility of this approach in an in vivo mouse model. When grafted keratinocytes that express MDR are topically selected with colchicine, long-term high-level expression of MDR is maintained in a significant percentage of cells. Quantitative PCR on the keratinocyte populations in the human skin grafts demonstrated that colchicine selected for keratinocyte stem cells expressing MDR, and prevented the gradual decline of genetically-modified MDR-expressing keratinocytes that occurs in the absence of colchicine. We have now used this selectable skin gene therapy model to obtain persistent high-level gene expression and systemic delivery of the atrial natriuretic peptide in a mouse model. ANP has both natriuretic or diuretic and vasorelaxant effects and generally counterbalances the effects of the renin-angiotensin-aldosterone system. We have developed bicistronic lentiviral vectors capable of expressing high levels of ANP and MDR, and these vectors have been used to transduce human keratinocytes and fibroblasts. Raft cultures or human skin equivalents have been constructed with human keratinocytes and fibroblasts that express high levels of ANP peptide. These human skin equivalents have been grafted onto immunocompromised mice and secrete human ANP systemically, with significant serum levels in grafted mice, and are able to normalize blood pressure in hypertensive mice and actually lower blood pressure in mice with normal blood pressure. This proof-of-concept success indicates that other therapeutic genes of interest can also be pursued with this approach.